Entrapped materials are substances that have some restriction in their ability to freely move (e.g., dissociate, dissolve, or diffuse) in an environment. A few examples of entrapped materials are a bioactive agent encapsulated in a microcapsule, a reactive agent coated onto a substrate, an enzyme covalently attached to a bead, or a macromolecule entangled in a gel or fiber matrix. Having a wide number of uses, such as controlled release systems, structural modifiers, and sensor or reactive materials, entrapped materials and methods for their preparation are an important field of research.
Typically, entrapped materials are formulated as membranes, coatings, or capsules. Current methods for forming such materials include emulsion polymerization, interfacial polymerization, dissolution, emulsification, gelation, spray-drying, vacuum coating, and adsorption onto porous particles. Common materials used in these methods include polymers, hydrocolloids, sugars, waxes, fats, metals, and metal oxides.
For the controlled release of entrapped liquid materials, the use of membranes, coatings, capsules, etc., is well known. For example, controlled-release materials have been used in the preparation of graphic arts materials, pharmaceuticals, food, and pesticide formulations. In agriculture, controlled-release techniques have improved the efficiency of herbicides, insecticides, fungicides, bactericides, and fertilizers. Non-agricultural uses include encapsulated dyes, inks, pharmaceuticals, flavoring agents, and fragrances.
The most common forms of controlled-release materials are coated droplets or microcapsules, coated solids, including both porous and non-porous particles, and coated aggregates of solid particles. In some instances, a water-soluble encapsulating film is desired, which releases the encapsulated material when the capsule is placed in contact with water. Other coatings are designed to release the entrapped material when the capsule is ruptured or degraded by external force. Still further coatings are porous in nature and release the entrapped material to the surrounding medium at a slow rate by diffusion through the pores.
Other materials have been formulated as emulsifiable concentrates by dissolving the materials in an organic solvent mixed with a surface-active agent or as an oily agent. In solid form, insecticides have been formulated as a wettable powder in which the insecticide is adsorbed onto finely powdered mineral matter or diatomaceous earth, as a dust or as granules.
Enzymes and proteins have become popular materials for entrapment. For example, enzyme entrapment on a solid support has been studied extensively as a simple means of protein stabilization and catalyst separation and recovery from reaction systems (Gemeiner, In Enzyme Engineering, Gemeiner, Ed., Ellis Horwood Series in Biochemistry and Biotechnology, Ellis Horwood Limited: West Sussex, England, 1992, pp 158-179; Mulder, Basic Principles of Membrane Technology, Kluwer Academic Publishers: Dordrecht, 1991). Entrapment of enzymes on solid supports can result in improved stability to pH and temperature and aid in separation of the enzyme from the reaction mixture, and also for formation of enzyme electrodes for sensor applications.
There are four principal methods available for immobilizing enzymes and other proteins: adsorption, covalent binding, entrapment, and membrane confinement. Typical materials used for these purposes include silica, polyaniline, acrylics, chitin, and cellulose (Gemeiner, In Enzyme Engineering, Gemeiner, Ed., Ellis Horwood Series in Biochemistry and Biotechnology, Ellis Horwood Limited: West Sussex, England, 1992, pp 158-179; Krajewska, Enz Microb Technol 2004, 35:126-139). Entrapment of enzymes within gels or fibers is typically used in processes involving low molecular weight substrates and products. Entrapment in calcium alginate is also used for immobilization of microbial, animal, and plant cells.
For entrapping proteins and other biomolecules, the use of cellulose, which is hydrophilic and wettable, can be desirable because it helps create a compatible environment as compared to hydrophobic materials (Tiller et al., Biotechnol Appl Biochem 1999, 30:155-162; Sakai, J Membr Sci 1994, 96:91-130). In addition, cellulose is robust, chemically inert under physiological conditions, and non-toxic, all of which are important for protein survival and advantageous for industrial processing. One method for enzyme immobilization uses polysaccharide activation in which cellulose beads are reacted under alkali conditions with cyanogen bromide. The intermediate produced is then covalently coupled with soluble enzymes. Enzymes can also be entrapped in cellulose acetate fibers by formulation of an emulsion of the enzyme plus cellulose acetate in dichloromethane, followed by extrusion of fibers.
In other examples, materials can be entrapped by dissolving and reconstituting cellulose. However, traditional cellulose dissolution processes, including the cuprammonium and xanthate processes, are often cumbersome or expensive and require the use of unusual solvents, typically with a high ionic strength and are used under relatively harsh conditions. (Kirk-Othmer, Encyclopedia of Chemical Technology, Fourth Edition 1993, Vol. 5, p. 476-563.) Such solvents include carbon disulfide, N-methylmorpholine-N-oxide (NMMNO), mixtures of N,N-dimethylacetamide and lithium chloride (DMAC/LiCl), dimethylimidazolone/LiCl, concentrated aqueous inorganic salt solutions (e.g., ZnCl/H2O, Ca(SCN)2/H2O), concentrated mineral acids (e.g., H2SO4/H3PO4), or molten salt hydrates (e.g., LiC4.3H2O, NaSCN/KSCN/LiSCN/H2O). These cellulose dissolution processes break the cellulose polymer backbone, resulting in regenerated products that contain an average of about 500 to about 600 glucose units per molecule rather than the native larger number of about 1500 or more glucose units per molecule. In addition, processes such as that used in rayon formation proceed via xanthate intermediates and tend to leave some residual derivatized (substituent groups bonded to) glucose residues, as in xanthate group-containing cellulose.
U.S. Pat. No. 5,792,399 discloses the use of N-methylmorpholine-N-oxide (NMMNO) solutions of cellulose to prepare regenerated cellulose that contained polyethyleneimine (PEI). That patent discloses that one should utilize a pre-treatment with the enzyme cellulase to lessen the molecular weight of the cellulose prior to dissolution. In addition, it discloses that NMMNO decomposes at the temperatures used for dissolution to provide N-methylmorpholine as a degradation product that could be steam distilled away from the cellulose solution. The presence of PEI is said to lessen the decomposition of the NMMNO.
Other processes that can provide a solubilized cellulose do so by forming a substituent that is intended to remain bonded to the cellulose, such as where cellulose esters like the acetate and butyrate esters are prepared, or where a carboxymethyl, methyl, ethyl, C2-C3 2-hydroxyalkyl (hydroxyethyl or hydroxypropyl), or the like group is added to the cellulose polymer. Such derivative (substituent) formation also usually leads to a lessening of the degree of cellulose polymerization so that the resulting product contains fewer glucose units per molecule than the cellulose from which it was prepared.
Against this background, many formulations of entrapped materials pose a variety of problems, such as the pollution of the environment caused by organic solvents used in the emulsions and by dust resulting from the wettable powders, and the costs associated with the removal of unwanted byproducts. Also, the use of cellulose in such compositions is generally associated with a number of drawbacks, most notably, the need for extensive chemical activation and functionalization necessary in order to attach biomolecules to the surface (Klemm et al., Comprehensive Cellulose Chemistry, Wiley VCH: Chichester, 1998; Vol. 2.; Chesney et al., Green Chem 2000, 2:57-62; Stöllner et al., Anal Biochem 2001, 304:157-165). Methods that involve cellulose solubilization can suffer from a break-down of the cellulose backbone, the requirement of exotic solvents and additional steps, and unwanted derivatization of the cellulose. Furthermore, for these formulations to have long-term residual effectiveness, an amount of entrapped material much higher than that used in normal applications can be required, and this increased amount can affect the environment or cause problems of safety.
Because the preparation of such entrapped materials can be technically difficult and environmentally harmful, there is a strong demand for methodologies that can reduce or simplify the entrapment process. There is also a need for superior formulations that can effectively replace the emulsifiable concentrates, interfacially-polymerized or wettable powders, and are safer to use. Also, the need for processes and formulations that allow for the utilization of cellulosic materials is highly desirable for a formulation that maintains a high degree of efficacy over long periods. Disclosed herein are compositions and methods that meet these and other needs.